Live bacteria, such as probiotic bacteria or live attenuated pathogens, represent attractive systems for the delivery of biologically active agents as they generally allow oral administration and the sustained release of the agent over a protracted period of time, eliminating the need for repeat doses.
As the therapeutic potential of bacterial delivery systems has been recognised a need to develop expression cassettes for the delivery of the active agents has also developed. These expression cassettes have mainly focused on the use of replicative plasmids; however, as these plasmids are essentially unstable ie often being lost by the bacterium over time, research to date has concentrated on plasmid stabilization. Most of the research in this area has been based on auxotrophy or gene essentiality using the expression in trans of the corresponding missing gene. For example, plasmid expression vectors have been developed harbouring the gene encoding aspartate β-semialdehyde dehydrogenase, an enzyme essential for the viability of bacteria (Galan et al., (1990), Gene, 94:29-35). These vectors were used in Salmonella vaccine strains harbouring lesions in the host asd gene encoding the enzyme. Thus, loss of the plasmid resulted in cell death and prevented selection of bacteria without the plasmid.
Accordingly, there remains a need for a delivery system that provides the stable, constitutive expression of vaccine antigens or biologically active molecules at sufficient levels to enable their use in clinical practice.